Team:Nevada/Notebook

From 2009.igem.org

Revision as of 21:47, 8 October 2009 by Khwajay (Talk | contribs)


Home The Team The Project Parts Submitted to the Registry Modeling Notebook


Contents

June 8, 2009 to June 15, 2009

Janice/Leigh:

  • Made LB agar plates and liquid LB with tetracycline.
  • Made tetracycline stock solution (5mg/ml).

Chris/Joey:

Tony/Nick:

  • RE digestion of RBS, Lac I, 2x Term, out of BioBrick Plasmids (2x Terminator digested with XbaI/SpeI, everything digested with NotI.)
  • Ran 0.8% Agarose gel of digestions above.

June 16, 2009 to June 23, 2009

Janice/Leigh:

  • Made LB liquid media and performed QIAGEN Plasmid Maxi Preps for
  1. BBa_B0014 in pSB1AK3 (double terminator)
  2. BBA_B0034 in pSB1A3 (ribosome binding site)
  3. BBA_R0011 in pSB1A3 (lac I promoter)
  4. BBa_I0500 in pSB2K3 (pBAD/Arac promoter).
  • Made glycerol stocks for 1) to 4).
  • Minipreps were done on the following Arabidopsis genes:
  1. cinnamoyl-CoA reductase (CCR2)
  2. cinnamoyl-CoA reductase (CCR1)
  3. phenylalanine-ammonia lyase
  4. 4-coumarate:CoA ligase 1
  5. 4-coumarate:CoA ligase 2
  • Digestions:
Cinnamoyl-CoA reductase (CCR2) was digested with HindIII and SalI.
Cinnamoyl-CoA reductase (CCR1) was digested with EcorI and HindIII.
Phenylalanine-ammonia lyase was digested with EcoRI and SacI.
4-coumarate:CoA ligase 1 was digested with HindIII.
4-coumarate:CoA ligase 2 was digested with EcoRI and HindIII.

Chris/Joey:

Prepare chemically competent DH4-alpha cells as described here: [http://openwetware.org/wiki/Preparing_chemically_competent_cells]

  • TSS buffer was prepared as described in aforementioned article

Tony/Nick:

  • PCR of CCR2 (cinamoyl CoA reductase gene)

Sheena:

June 24, 2009 to June 30, 2009

Janice/Leigh:

  • BBa_J04450 in plasmid pSB1AT3 (RFP), BBa_J04450 in plasmid pSB1AT3 (ccdB), BBa_I52001 in plasmid pSB3T5 (ccdb), BBa_J04450 in plasmid pSB3T5 (RFP) were transformed into NEB10 chemically competent cells. These are the tetracycline resistant plasmid backbones.
  • Cultures were grown and DNA was extracted for the plasmid backbones described above.

Chris/Joey:

Tony/Nick:

  • Topo clone CCR2, tranformed into One Shot, and spread on LB+Amp plates.
  • Isolated colonies from transformation and spread on fresh LB+Amp plates.
  • Minipreped transformed colonies from fresh plate.
  • RE digestion of Minipreped CCR2 and run on 0.8% Agarose gel.
  • RE digestion of CCR2, CCR1(another Cinamoyl CoA reductase), Phenyalanine Lyase (PAL1) and Ran Gel.

July 1, 2009 to July 7, 2009

Janice/Leigh:

  • The Arabidopsis genes were digested and were analyzed by gel electrophoresis.
  • The double terminator, ribosome binding site, lac I promoter, pBAD promoter, and two tetracycline resistant plasmid backbones were restriction digested and ran on an agarose gel.

Chris/Joey:

Tony/Nick:

  • RE digestion of CCR2 with XbaI, EcoRI, PstI, SpeI (separate digests).
  • Sequenced CCR2.

July 8, 2009 to July 15, 2009

Janice/Leigh:

  • Performed the 3-way ligation for the lac I promoter (upstream part), ribosome binding site (RBS) (downstream part), and the destination plasmid (BBa_I52001 in pSB3C5; chloramphenicol resistance).
  1. Digestion: lac I promoter was digested with EcoRI and SpeI; RBS was digested with XbaI and PstI; destination plasmid was digested with EcoRI and PstI. All were digested at 37ºC for one hour and the restriction enzymes were deactivated at 80ºC for 20 minutes.
  2. Ligation: The lac I promoter, RBS, and the destination plasmid (BBa_I52001 in pSB3C5) were ligated and incubated for one hour.
  • The 3-way ligation was transformed into NEB10 competent cells.
    • 20 μl of the 3-way ligation described above were added to 100 μl of NEB10 competent cells.
    • Chloramphenicol was spread onto the LB plates to a final concentration of 25 μg/ml.
    • 10, 100, and 160 μl of the transformation were added to 3 LB plates.
    • 80 colonies of the 3-way ligation were screened on Amp and Chloramphenicol plates, resulting with 6 colonies that grew only on Chloramphenicol.

Chris/Joey:

Tony/Nick:

  • PCR CCR2 and ran on gel.

July 16, 2009 to July, 23 2009

Janice/Leigh:

  • Screening of the 3-way ligation (lac promoter/RBS/backbone) colonies
    • Six selected colonies were cultured in LB-Chloramphenicol (25μg/ml)
    • The DNA was collected using a QIAGEN miniprep kit and concentration was determined by the Nanodrop.
    • All six colonies were digested with EcoRI to linearize, expecting a fragment length of 2.7kb.
    • Digests were run on a 1% agarose gel along with uncut DNA. Five of the six colonies resulted in the correct band length.
  • 3-way ligation of RBS (BBa_R0011), pBAD promoter (BBa_I0500), and Chloramphenicol backbone (pSB3C5)
    • The upstream part, pBAD promoter, was digested with EcoRI and SpeI
    • The downstream part, RBS, was digested with XbaI and PstI
    • The Chloramphenicol bakcbone was digested with EcoRI and PstI
    • All digests were incubated for one hour at 37C followed by a 20 minute deactivation step at 80C
    • 4μl of each of the three digests were used in a final 20μl ligation reaction with a one hour incubation at root temperature, followed by a 20 minute deactivation step at 80C
    • The ligation was transformed into NEB10 cells and volumes of 10, 50, 100μl, and the rest was spread on LB plates containing Chloramphenicol (25μg/ml)
    • The plates were incubated overnight at 37C

Chris/Joey:

Tony/Nick:

  • Topo cloned CCR2 and transformed into NEB 10 Cells.
  • Plated Topo clones on Xgal/Amp plates.
  • Streaked selected colonies on Amp plates.
  • Minipreped and RE Digestion of CCR2 Topo clones.

July 24, 2009 to July 31, 2009

Janice/Leigh:

  • Screening of the 3-way ligation (pBAD promoter/RBS/backbone) colonies
    • 7 colonies were selected and cultured in 3ml of LB-Chloramphenicol (25μg/ml)
    • Each colony was digested with PstI to linearize and a double digest of PstI/EcoRI to cut out the pBAD promoter(1.2kb)
    • The digests were run on a 1% agarose gel, none of which resulted with the expected fragments lengths.
  • Sequencing Results of the 3-way ligation (lac promoter/RBS/backbone)
      • Two colonies which resulted with the correct fragment length from the restriction digests were sent to be sequenced to the Nevada Genomics Center using the Biobrick primers VF2 and VR. 500ng of each of the colonies was used for each of the sequencing reactions

Chris/Joey:

Tony/Nick:

  • 3 Way ligation of CCR2 with 2x terminator, and (BBa_52001) CPC resistant Backbone.
  • Transformation of 3 Way into NEB 10.

August 1, 2009 to August 8, 2009

Janice/Leigh:

  • Repeated the 3-way ligation for the lac I promoter (upstream part), ribosome binding site (RBS) (downstream part), and the destination plasmid (BBa_I52001 in pSB3C5; chloramphenicol resistance).
  1. Digestion: lac I promoter was digested with EcoRI and SpeI; RBS was digested with XbaI and PstI; destination plasmid was digested with EcoRI and PstI. All were digested at 37ºC for one hour and the restriction enzymes were deactivated at 80ºC for 20 minutes.
  2. Ligation: The lac I promoter, RBS, and the destination plasmid (BBa_I52001 in pSB3C5) were ligated and incubated for one hour.
  • The 3-way ligation was transformed into NEB10 competent cells.
    • 20 μl of the 3-way ligation described above were added to 100 μl of NEB10 competent cells.
    • Chloramphenicol was spread onto the LB plates to a final concentration of 25 μg/ml.
    • 10, 100, and 160 μl of the transformation were added to 3 LB plates.
    • 80 colonies of the 3-way ligation were screened on Amp and Chloramphenicol plates, resulting with 6 colonies that grew only on Chloramphenicol.
  • Sequencing results resulted that the lac I promoter had mutated and was not the complete sequence
  • Re-transformation of Lac I promoter (BBa_R0011) in NEB10 company competent cells
    • selected one colony and cultured in 3ml of LB-Amp
    • DNA of the lac I promoter was collected using the QIAGEN miniprep kit

Chris/Joey:

Tony/Nick:

  • Streaked colonies from CCR2 + 2x term + I52001 CPC onto LB+CPC plates.
  • Selected colonies and Minipreped them.
  • Repeat of 3-way ligation, selection, and miniprep.


August 9, 2009 to August 16, 2009

Janice/Leigh:

  • Made NEB10 competent cells
    • Checked for competency by transforming the cells with 100ng of PUC
  • Sent the chosen Lac I promoter (BBa_R0011) in for sequencing to the Nevada Genomics Center. The promoter was sequenced in both directions using Biobrick primers VF2 and VR.
    • Sequencing results confirmed that BBa_R0011 contains the complete lac I promoter sequence
    • Cultures set for the promoter in LB-Amp and DNA was collected using a QIAGEN miniprep kit
  • 3-way ligation of lac I promoter (BBa_R0011), RBS (BBa_B0034), and destination plasmid pSB3C5
    • The upstream part, lac I promoter, was digested with EcoRI and SpeI
    • The downstream part, RBS, was digested with XbaI and PstI
    • The Chloramphenicol bakcbone was digested with EcoRI and PstI
    • All digests were incubated for one hour at 37C followed by a 20 minute deactivation step at 80C
    • 4μl of each of the three digests were used in a final 20μl ligation reaction with a one hour incubation at root temperature, followed by a 20 minute deactivation step at 80C
    • The ligation was transformed into NEB10 cells and volumes of 10, 50, 100μl, and the rest was spread on LB plates containing Chloramphenicol (25μg/ml)
    • The plates were incubated overnight at 37C

Chris/Joey:

Tony/Nick:

  • Minipreps from 3 way ligation (CCR2+2xTerm+I52001)ran on 1% agarose gel.
  • RE Digestion of 3 way to confirm insert
  • 3 way sequenced

August 17, 2009 to August 24, 2009

Janice/Leigh:

  • Screening of the 3-way ligation (lac promoter/RBS/backbone) colonies
    • 62 colonies were replica plated on LB-AMP and LB-chloramphenicol
    • Four selected colonies that only grew on LB-chloramphenicol were cultured in LB-Chloramphenicol (25μg/ml)
    • The DNA was collected using a QIAGEN miniprep kit and concentration was determined by the Nanodrop.
    • All six colonies were digested with EcoRI to linearize, expecting a fragment length of 2.7kb.
    • Digests were run on a 1% agarose gel along with uncut DNA. Three for the four colonies resulted in the correct band length.
    • DNA from two of the colonies was sent for sequencing at the Nevada Genomics center. Both colonies were sequenced in two directions using the Biobrick primers VF2 and VR.


Chris/Joey:

Tony/Nick:

  • 4-coumarate: CoA Ligase (Gene 2) minipreped.
  • Gene 2 RE Digested to linearlize (SalI).

August 25, 2009 to September 1, 2009

Janice/Leigh:

  • Sequencing results of the 3-way ligation (lac/RBS/chloramphenicol backbone) resulted with the correct sequence
    • Bulk-up DNA of the 3-way ligation using the I, II, III miniprep protocol
  • Transformation of mutated AT4CL2 (4-Coumarate:CoA ligase 2) sent from Dr. Kombrink into DH5α

Chris/Joey:

Tony/Nick:

  • PCR pf Gene 2.

September 2, 2009 to September 9, 2009

Janice/Leigh:

  • Selected colonies of the AT4CL2 transformation were cultured in LB-Amp
    • DNA was collected using the QIAGEN miniprep kit
    • DNA was digested with PstI and Sal I
  • 3-way ligation of final construct (lac I promoter/RBS/Gene 3/terminator)
    • 500 ng of the upstream part, lac/RBS 3-way ligation, was digested with EcoRI and SpeI
    • 500ng of the downstream part, Gene 3/terminator 3-way ligation, was digested with XbaI and PstI
    • 500 ng of the high copy tetracylcine resistant backbone (pSB1AT3) was digested with EcoRI and PstI
    • All digest were incubated for an hour at 37C and deactivated for 20 minutes at 80C
    • 4μl of each of the digests was used for the ligation reaction
    • the ligation reaction was incubated at room temperature for 1 hour and deactivated for 20 minutes at 80C
    • The ligation was transformed into NEB10 competent cells and plated on LB-Tet


Chris/Joey:

Tony/Nick:

  • 3 way ligation of (gene 2 + Lac/RBS + I52001 CPC BB)
  • 3 way ligation of (gene 3 + Lac/RBS + I52001 Tet BB)
  • 2 way ligation of (Gene 2 + I52001 CPC BB)


September 10, 2009 to September 17, 2009

Janice/Leigh:

  • Screening of the colonies from the 3-way final construct (promoter/RBS/Gene3/terminator)
    • Colonies were replica plated on LB-tetracycline and LB-chloramphenicol plates
    • Four of the six colonies grew only on the LB-tetracycline plates
    • The four colonies were digested with SalI and the digests were run on a 1% agarose gel
    • None of the digests resulted with the correct fragment lengths
  • Redo of the 3-way ligation final construct (promoter/RBS/Gene3/terminator)
    • Six colonies were replica plated on LB-tetracylcine and LB-chloramphenicol plates
    • Four colonies grew only on LB-tetracycline
    • DNA of the four colonies was collected using the QIAGEN miniprep kit
    • DNA was digested with EcoRI and run on a 1% agarose gel
    • DNA from four colonies was sent for sequencing to the Nevada Genomics Center using the Biobrick primers, VF2 and VR.

Chris/Joey:

Tony/Nick:

  • Transformation of 3 way ligations, and 2 way ligation above into NEB 10.
  • RE Digestion of 3 way ligations, and 2 way ligation and ran on 1% agarose gel

September 18, 2009 to September 25, 2009

Janice/Leigh:

  • Sequencing results returned did not contain the sequence of the final construct


Chris/Joey:

Tony/Nick:

September 26, 2009 to October 3, 2009

Janice/Leigh:

  • Re-do of the 3-way final construct: promoter/RBS/Gene 3/terminator
    • New Tetracycline stock and LB-tetracycline plates were prepared
    • Two colonies grew
  • Cultures were set for high-copy tetracycline backbone, low-copy tetracycline backbone, the two final construct colonies and the lac/RBS
    • DNA was digested and run on a 1% agarose gel
    • The two colonies of the final construct did not result with the correct size

Chris/Joey:

Tony/Nick:

  • Minipreped 3 way ligations above, RE digested, and ran on 1% agarose Gel

October 4, 2009 to October 11, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

  • 3 way ligation of (Gene 2 + RBS + I52001 Tet BackBone.)
  • Minipreped Janice and Leigh's Final Construct of (LacI/RBS + Gene 3 + 2x Term + I52001 TET BB).
  • RE Digestion (EcoRI/PstI) of (Gene 2 + 2x term + I52001 CPC BB).
  • RE Digestion (EcoRI/PstI) of (Gene 2 + RBS + I52001 Tet BB).

October 12, 2009 to October 19, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

October 20, 2009 to October 27, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

October 28, 2009 to November 4, 2009

Janice/Leigh:

Chris/Joey:

Tony/Nick:

Home The Team The Project Parts Submitted to the Registry Modeling Notebook
Retrieved from "http://2009.igem.org/Team:Nevada/Notebook"